The Mycobacterium avium complex (MAC) is the most significant of the environmental mycobacteria that cause disease in susceptible humans. Most MAC infections are thought to come from stably colonized drinking water supplies. The predominant way of life for bacteria in water is growth in surface-adherent biofilms, and recent studies have shown that MAC is no exception. It is present in vast numbers in biofilm samples taken from water distribution systems, and it appears to be the dominant Mycobacterium species in such environments. Genetic approaches have been used to analyze biofilm formation by the fast-growing model species M. smegmatis; however the genetics of biofilm formation by MAC remains to be explored. Using newly developed genetic and genomic tools, combined with authentic laboratory models of MAC biofilm development, we will conduct the first such investigation. The specific aims of this exploratory project are 1) to identify MAC genes involved in the early stages of biofilm formation by using a novel "transposome" mutagenesis approach, and 2) to identify MAC genes involved in the formation of mature two-species biofilms by using signature-tagged mutagenesis and transposon site hybridization. By establishing methods and conditions for genetic analysis of biofilm formation by MAC, we will begin to shed light on an aspect of mycobacterial biology that is almost entirely uncharacterized. The results will improve our understanding of the microbial ecology of environmental mycobacteria, and they may lead to improved methods for protecting susceptible people from MAC infection.